Glycosyltransferases catalyze transfer of a monosaccharide residue from a sugar nucleotide (sugar donor) to the non-reducing terminus of a specific sugar acceptor. This process occurs within cells, especially in the Golgi apparatus, and is involved in the synthesis of all of the known complex carbohydrates, including glycoproteins, glycolipids and glycosaminoglycans. The reaction is represented in FIG. 1, in which different monosaccharides are represented by different shapes. (Taken from Roth, S. et al., Cell and Tissue Interactions, pp 209-223, ed. J. W. Lash and M. M. Burger, Raven Press, 1977.)
FIG. 1 depicts a trisaccharide sugar acceptor linked at its reducing end to a protein or lipid moiety, which is represented by a wavy line. A glycosyltransferase, represented as "enzyme", catalyzes the transfer of a monosaccharide from a glycosyl donor (sugar-nucleotide phosphate donor) to the nonreducing terminus of the sugar acceptor. This reaction involves a cofactor, which is generally a divalent cation (e.g., Mn.sup.2+) In the reaction products are a tetrasaccharide and a free nucleotide.
Such reactions are carried out within cells to effect the addition of monosaccharides to a glycosylated (carbohydrate-containing) substrate, such as glycoproteins, glycolipids, and proteoglycans. The reactions occur in the Golgi apparatus and are catalyzed or directed by a glycosyltransferase which is specific not only for the sugar-nucleotide substrate (for the monosaccharide to be transferred), but also for the specific carbon atom of the sugar or amino acid acceptor involved. For example, one glycosyltransferase catalyzes the transfer of N-acetylneuraminic acid (sialic acid) from CMP-sialic acid only to the 3 carbon atom galactose and a second catalyzes transfer only to the 6 carbon atom. Darnell, J. et al., Molecular Cell Biology, p. 958, Scientific American Books (1986). These transferases are named according to the sugar donors they utilize. For example, all galactosyltransferases transfer galactose from uridine diphosphate galactose to their specific acceptors, while neuraminyltransferase (sialyltransferase) transfers N-acetyl neuraminic acid (sialic acid) from its cytidine monophosphate derivative (CMP-Neu) to the required acceptor. As far as is known, no transferase can use more than one type of sugar donor. B. D. Shur and S. Roth, Biochim. Biophys. Acta 415:473-512 (1975).
Recently, it has been found that glycosyltransferases are present on cell surfaces, as well as within cell organelles, such as Golgi apparatus, endoplasmic reticula and mitochondrial membranes. B. D. Shur and S. Roth, Biochim. and Biophy. Acta 415:473-512 (1975) and references cited therein.
Evidence suggests that on cell surfaces, glycosyltransferases participate in a myriad of cellular interactions by binding their specific carbohydrate substrates on adjacent cells or in the extracellular matrix. It has been shown that if a cell surface glycosyltransferase molecule comes in contact with an appropriate acceptor (e.g., a glycoprotein) on another cell, the glycosyltransferase will bond non-catalytically with the acceptor. Thus, there will be an initial adhesive recognition as the result of creation of a transferase-substrate complex. In the extracellular environment, there are no sugar-nucleotides and cofactor concentrations are well below those needed for glycosyltransferase activity; thus, there is no enzymatic activity. Addition of the appropriate sugar-nucleotide and co-factor at appropriate levels has been shown to result in enzymatic addition of monosaccharide to acceptor (glycosylated substrate); glycosyltransferases present cease to act as cell adhesion molecules. Surface glycosyltransferases play a role in embryonic cell adhesion and migration, embryogenesis, immune recognition, growth control B. S. Shur, Mol. Cell. Biochem. 61:143-158 (1984).
For example, galactosyltransferase in sperm heads, which catalyzes the transfer of galactose from uridine 5'-diphosphate galactose to terminal N-acetylglucosamine residues, may recognize and bind to specific N-acetylglucosamine residues on the egg zona pellucida. P. M. Wassarman, Science, 235:553 (1987). Recognition and binding are accomplished through formation of an enzyme-substrate complex.
Presently, assessment of glycosyltransferase activity or function typically relies on use of sugar-nucleotides which are isotopically labeled at the monosaccharide (e.g., .sup.3 H or .sup.14 C) or monitoring of the release of hydrolyzed nucleotide spectrophotometrically. For example, most studies of cell surface glycosyltransferase activity rely on the addition of sugar nucleotides that are isotopically labeled at the monosaccharide. See Schwyzer, M. and R. L. Hill, J. Biol. Chem., 252:2338-2345 (1977). However, assays of this kind must control for the potential intracellular utilization of free labeled sugars which result from nucleotide hydrolysis by sugar phosphatase and nucleotide pyrophosphatases. Shur, D. B., Mol. Cell. Biochem. 61:143-158 (1984). These radioassays, in addition, are cumbersome and time-consuming.
Immunometric methods have also been developed for localization of cell surface glycosyltransferases. Anti-glycosyltransferase antibodies directed against the soluble form of the enzyme have been used to localize cell surface glycosyltransferase activity. However, use of these antibodies is limited because the anti glycosyltransferases are not very pure and their use generally disrupts the cell adhesion functions of glycosyltransferase. Phototungstic acid has also been used to visualize and localize glycosyltransferases. However, it can be used only with high molecular weight acceptor molecules and works poorly with the acid labile sialytransferases. See Schachter, H. and S. Roseman, "Mammalian Glycosyltransferases", pp 85-160 in: The Biochemistry of Glycoproteins and Proteoglycans, (ed. W. J. Lennarz), Plenum Press, 1980.
It would be useful to have a method of detecting glycosyltransferases and/or monitoring their activity which does not rely on the use of radio isotopes. Such a method would be particularly valuable if it made detection and/or monitoring of such enzymes in living cells.